Properties of a collagenolytic enzyme from Bipalium kewense

A collagenolytic enzyme from the land planarian Bipalium kewense has been purified by preparative isoelectric focusing. The enzyme has a molecular weight of 47,000 +/- 2,000 and appears to be dimeric. It has an isoelectric point of 4.6 +/- 0.1 and a high content of acidic amino acids. The amino acid composition of the Bipalium collagenase is similar to that of human skin fibroblast collagenases but clearly different from previously reported collagenolytic proteases from other invertebrates, Uca pugilator and Hypoderma lineatum. In its action on guinea-pig collagen, the enzyme produces distinct products, at low incubation temperatures, different from those produced by vertebrate and other invertebrate collagenolytic enzymes. These products have glycine as their N-terminal amino acids. As determined by viscosity measurements, the Bipalium collagenase is more active on invertebrate, earthworm, collagen than it is on the vertebrate, Type I guinea-pig skin, collagen. The Bipalium collagenase differs from both bacterial and vertebrate collagenases as well as from invertebrate, collagenolytic serine proteases.     

The isolation and properties of collagenolytic proteases from crab hepatopancreas

A mixture of collagenolytic proteases has been isolated from the Kamchatka crab hepatopancreas. The four individual enzymes were further separated with FPLC and partially characterized. Crab collagenolytic proteases possess a high activity against different types of collagen, especially against calf skin collagen Type III and bovine lens capsule collagen Type IV, which is resistant to the microbial Clostridium sp. collagenases. In contrast with microbial collagenases the crab enzymes are good general proteases, able to cleave standard synthetic and protein substrates and possess a chymotrypsin-, trypsin- and elastase-like specificity. N-Terminal sequence analysis revealed that crab collagenolytic proteases had evolved from a trypsin-like ancestor. Crab proteases, structurally belonging to the trypsin-like enzymes, nevertheless, possess the unique ability, among this class of enzymes, to cleave the native insoluble collagen. It seems that crab collagenolytic proteases and true metalloenzyme vertebrate and microbial collagenases have certain common structural features particularly in the regions of their substrate binding site.     

Degradation of collagen substrates by a trypsin-like serine protease from the fiddler crab Uca pugilator

The collagenolytic properties of a trypsin-like protease from the hepatopancreas of the fiddler crab Uca pugilator have been examined. All collagen types, I-V, were attacked by this enzyme. Types III and IV were degraded much more rapidly than types I, II, and V. Crab protease produced multiple cleavages in the triple helix of each collagen at 25 degrees C; only in the case of type III collagen, however, was a major cleavage observed at a 3/4:1/4 locus that corresponded to the region of collagen susceptibility to mammalian collagenase action. Additionally, both the affinity and the specific activity of the crab protease for native collagen were lower than those which characterize mammalian collagenase. The results of this study, in conjunction with a previous report on the collagenolytic activity of another serine protease from the fiddler crab [Welgus, H. G., Grant, G. A., Jeffrey, J. J., & Eisen, A. Z. (1982) Biochemistry 21, 5183], suggest that the following properties distinguish the action of these invertebrate collagenolytic enzymes from the metalloenzyme collagenases of mammals: (1) broad substrate specificity, including both noncollagenous proteins and collagen types I-V; (2) ability to cleave the native triple helix of collagen at multiple loci; (3) reduced affinity or higher Km for collagen; and (4) lower specific activity on collagen fibrils.     

Collagenolytic Activity of Some Marine Bacteria

Reconstituted, acid-extracted collagen was used to prepare a medium to screen proteolytic marine bacteria for their ability to elaborate collagenolytic enzymes. The medium was resistant to solubilization by trypsin, hyaluronidase, chondroitinase ABC, and various marine proteinases, but was readily hydrolyzed by commercial Clostridium collagenases. Eighty-seven marine isolates collected in the vicinity of Bermuda, Oahu (Hawaii), and Stone Harbor and Cape May, N. J., were screened. Approximately 44 per cent of the isolates were capable of elaborating enzymes that hydrolyzed reconstituted collagen gels. Several cultures produced collagenolytic enzymes only when grown in the presence of collagen or degradation products of collagen, and with very few exceptions the presence of collagen in the medium greatly enhanced collagenolytic enzyme production. The enzymes from a collagenolytic Bermuda marine isolate were studied in more detail to illustrate that the enzymes capable of hydrolyzing reconstituted collagen were separable from nonspecific proteinases by zone electrophoresis and that these enzymes were true collagenases by virtue of their ability to hydrolyze native bovine Achilles'tendon obtained from three different sources.     

Bovine spleen cathepsin B1 and collagenolytic cathepsin. A comparative study of the properties of the two enzymes in the degradation of native collagen.

Bovine spleen cathepsin B1 and collagenolytic cathepsin were separated by chromatography on Amberlite IRC-50 and collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50). 2. Collagenolytic cathepsin degraded insoluble tendon collagen maximally at pH 3.5 and 28 degrees C; mainly alpha-chain components were released into solution. At 28 degrees C the telopeptides in soluble skin collagen were also cleaved to yield alpha-chain components. Collagenolytic cathepsin was thus similar to cathepsin B1 in its action against native collagen, but mixtures of these two enzymes exhibited a synergistic effect. 3. The addition of thiol-blocking compounds produced similar inhibition of collagenolytic cathepsin and cathepsin B1. The enzyme responded similarly to all other compounds tested except to 6-aminohexanoic acid, when collagenolytic cathepsin was slightly activated and cathepsin B1 was almost unaffected. 4. Leupeptin, which is a structural analogue of arginine-containing synthetic substrates, inhibited collagenolytic cathepsin as effectively as cathepsin B1. Collagenolytic cathepsin was shown to retain a low residual activity against alpha-N-benzoyl-DL-arginine p-nitroanilide during purification which was equivalent to 0.2% of the activity of cathepsin B1. 5. Cathepsin B1 and collagenolytic cathepsin could not be separated by affinity chromatography on organomercurial-Sepharose 4B. The two enzymes could be resolved on DEAE-Sephadex (A-50) and by isoelectric focusing in an Ampholine pH gradient. The pI of the major cathepsin B1 isoenzyme was 4.9 and the pI of collagenolytic cathepsin was 6.4. 6. From chromatography on Sephadex G-75 (superfine grade) the molecular weights were calculated to be 26000 for cathepsin B1 and 20000 for collagenolytic cathepsin. The difference in molecular weight was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis.     

Pulmonary fibroblasts mobilize the membrane-tethered matrix metalloprotease, MT1-MMP, to destructively remodel and invade interstitial type I collagen barriers

In acute and chronic lung disease, widespread disruption of tissue architecture underlies compromised pulmonary function. Pulmonary fibroblasts have been implicated as critical effectors of tissue-destructive extracellular matrix (ECM) remodeling by mobilizing a spectrum of proteolytic enzymes. Although efforts to date have focused on the catabolism of type I collagen, the predominant component of the lung interstitial matrix, the key collagenolytic enzymes employed by pulmonary fibroblasts remain unidentified. Herein, membrane type-1 matrix metalloprotease (MT1-MMP) is identified as the dominant and direct-acting protease responsible for the type I collagenolytic activity mediated by both mouse and human pulmonary fibroblasts. Furthermore, MT1-MMP is shown to be essential for pulmonary fibroblast migration within three-dimensional (3-D) hydrogels of cross-linked type I collagen that recapitulate ECM barriers encountered in the in vivo environment. Together, these findings demonstrate that MT1-MMP serves as a key effector of type I collagenolytic activity in pulmonary fibroblasts and earmark this pericellular collagenase as a potential target for therapeutic intervention.     

The action of cathepsin B and collagenolytic cathepsin in the degradation of collagen.

Cathepsin B and collagenolytic cathepsin were obtained from bovine spleen and human placenta and identified as thiol proteinases. Both enzymes degraded insoluble fibrous collagen maximally at pH 3.5 and soluble monomeric collagen near pH 4.5. The response to activators and inhibitors was similar for both enzymes. Collagenolytic cathepsin was unable to degrade the synthetic substrates of cathepsin B and was also shown to differ in its physico-chemical properties. Minor differences were noted in the action of these cathepsins on insoluble fibrous collagen from different tissues. It was concluded that the rate and extent of the dissolution of fibrous collagen was determined by the number and location of the interchain cross-links, the amount of the associated non-collagenous components and the type of solvent ions, but not by the collagen phenotype.     

A polyphasic approach to the exploration of collagenolytic activity in the bacterial community associated with the marine sponge Cymbastela concentrica

Collagen is an important, extracellular structural protein for metazoans and provides a rich nutrient source for bacteria that possess collagen-degrading enzymes. In a symbiotic host system, collagen degradation could benefit the bacteria, but would be harmful for the eukaryotic host. Using a polyphasic approach, we investigated the presence of collagenolytic activity in the bacterial community hosted by the marine sponge Cymbastela concentrica. Functional screening for collagenase activity using metagenomic library clones (227 Mbp) and cultured isolates of sponge's bacterial community, as well as bioinformatic analysis of metagenomic shotgun-sequencing data (106,679 predicted genes) were used. The results show that the abundant members of the bacterial community contain very few genes encoding for collagenolytic enzymes, while some low-abundance sponge isolates possess collagenolytic activities. These findings indicate that collagen is not a preferred nutrient source for the majority of the members of the bacterial community associated with healthy C. concentrica, and that some low-abundance bacteria have collagenase activities that have the potential to harm the sponge through tissue degradation.     

Characterisation of cathepsin B and collagenolytic cathepsin from human placenta

1. Human placental cathepsin B and collagenolytic cathepsin were separated by chromatography on columns of Amberlite CG-50. Collagenolytic cathepsin was partially purified by chromatography on DEAE-Sephadex (A-50) and Sephadex G-100. Cathepsin B was purified by chromatography on CM-cellulose and Sephadex G-100. 2. Both enzymes required activation by thiol compounds and were bound to organomercurial-Sepharose-4B. Sulphydryl-blocking reagents were inhibitory, which confirmed an essential thiol group to be present. 3. The enzymes degraded soluble calf skin collagen and insoluble bovine tendon collagen in the telopeptide region at pH 3.5 and 28 degrees C to yield mainly alpha-chain components. 4. In contrast to cathepsin B, collagenolytic cathepsin was found not to hydrolyse any of the low-molecular-weight synthetic substrates that were tested. 5. Leupeptin, a structural analogue of arginine-containing synthetic substrates, and antipain, an inhibitor of papain, were strongly inhibitory to both enzymes. 6. The isoelectric points of the enzymes were similar, being 5.4 for cathepsin B and 5.1 for collagenolytic cathepsin. 7. From chromatography on Sephadex G-100 the molecular weight of cathepsin B was calculated to be 24 500 and that of collagenolytic cathepsin to be 34 600.     

Resorption of endometrial collagen in oophorectomized rats

Uteri of rats that had been either oophorectomized or given a single dose of 17 beta-diethylstilbestrol or progesterone were studied as to the ultrastructural features of collagen resorption by the fibroblasts of the endometrial stroma. Electron microscopy showed several cytoplasmic vesicles within fibroblasts containing collagenous fibrils in various stages of breakdown. These findings suggest that fibroblasts have a collagenolytic action. Hence, this cell type may play a dual role in the metabolism of endometrial collagen - firstly being responsible for its synthesis, and secondly participating in its resorption.     

Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase

Inhibitory effect of green tea polyphenols viz., catechin and epigallocatechin gallate (EGCG) on the action of collagenase against collagen has been probed in this study. Catechin and EGCG treated collagen exhibited 56 and 95% resistance, respectively, against collagenolytic hydrolysis by collagenase. Whereas direct interaction of catechin and EGCG with collagenase exhibited 70 and 88% inhibition, respectively, to collagenolytic activity of collagenase against collagen and the inhibition was found to be concentration dependent. The kinetics of inhibition of collagenase by catechin and EGCG has been deduced from the extent of hydrolysis of (2-furanacryloyl-L-leucyl-glycyl-L-prolyl-L-alanine), FALGPA. Both catechin and EGCG exhibited competitive mode of inhibition against collagenase. The change in the secondary structure of collagenase on treatment with catechin and EGCG has been monitored using circular dichroism spectropolarimeter. CD spectral studies showed significant changes in the secondary structure of collagenase on treatment with higher concentration of catechin and EGCG. Higher inhibition of EGCG compared to catechin has been attributed to the ability of EGCG to exhibit better hydrogen bonding and hydrophobic interaction with collagenase.     

Structural and mechanistic insights into collagen degradation by a bacterial collagenolytic serine protease in the subtilisin family

A number of proteases in the subtilisin family derived from environmental or pathogenic microorganisms have been reported to be collagenolytic serine proteases. However, their collagen degradation mechanisms remain unclear. Here, the degradation mechanism of type I collagen fibres by the S8 collagenolytic protease MCP‐01, from Pseudoalteromonas sp. SM9913, was studied. Atomic force microscopy observation and biochemical analysis confirmed that MCP‐01 progressively released single fibrils from collagen fibres and released collagen monomers from fibrils mainly by hydrolysing proteoglycans and telopeptides in the collagen fibres. Structural and mutational analyses indicated that an enlarged substrate‐binding pocket, mainly composed of loops 7, 9 and 11, is necessary for collagen recognition and that the acidic and aromatic residues on these loops form a negatively charged, hydrophobic environment for collagen binding. MCP‐01 displayed a non‐strict preference for peptide bonds with Pro or basic residues at the P1 site and/or Gly at the P1’ site in collagen. His211 is a key residue for the P1‐basic‐residue preference of MCP‐01. Our study gives structural and mechanistic insights into collagen degradation of the S8 collagenolytic protease, which is helpful in developing therapeutics for diseases with S8 collagenolytic proteases as pathogenic factors and in studying environmental organic nitrogen degradation mechanisms.     

Levels of collagenolytic activity, β-glucuronidase,and collagen prolyl hydroxylase in paws from rats with developing adjuvant arthritis

The collagenolytic activity associated with insoluble collagen fibers separated from homogenates of inflamed paws from rats with adjuvant arthritis was quantitated using EDTA-sensitive solubilization of hydroxyproline as a measure of activity. Approximately 60% of the solubilized hydroxyproline was associated with dialyzable products. The level of collagenolytic activity in the paws increased with time after the induction of adjuvant arthritis and paralleled to a large extent the development of inflammation in both the adjuvant injected (right) hind paw and in the non-injected, contralateral paw. By day 26, the level of free collagenolytic activity in the injected paw had increased to a level 30 times normal while that in the contralateral paw had increased to a level 10 times normal. Treatment of the residues from the injected paws with trypsin resulted in the activation of a latent collagenolytic activity which, on day 26, accounted for approximately 50% of the total activity. The elevated level of collagen prolyl hydroxylase in the inflamed paw suggested that the rate of collagen synthesis was also increased. The activity of β-glucuronidase increased in the inflamed paw with time after the induction of adjuvant arthritis while that of cathepsin G was elevated as compared to normal in paws removed, 5 but not 22 days after the induction of adjuvant arthritis. The inflamed paw of the adjuvant rat may represent a useful system in which to study the role of collagenolytic enzymes in the destruction of connective tissue by inflammatory lesions.     

A collagenolytic serine protease with trypsin-like specificity from the fiddler crab Uca pugilator

A second collagenolytic serine protease has been isolated from the hepatopancreas of the fiddler crab, Uca pugilator. This enzyme cleaves the native triple helix of collagen under physiological conditions of pH, temperature, and ionic strength. In addition to its collagenolytic activity, the enzyme exhibits endopeptidase activity toward other polypeptides and small molecular weight synthetic substrates. The polypeptide bond specificity of this enzyme is similar to that of bovine trypsin as is its interaction with specific protease inhibitors. The amino-terminal sequence of this enzyme displays significant homology with other serine proteases, most notably with that of crayfish trypsin, and demonstrates that this enzyme is a member of the trypsin family of serine endopeptidases. The relatively unique action of this protease with regard to both collagenous and noncollagenous substrates has important implications concerning the specificity and mechanism of collagen degradation.     

Tumor-associated trypsinogen-2 (trypsinogen-2) activates procollagenases (MMP-1, -8, -13) and stromelysin-1 (MMP-3) and degrades type I collagen

A critical step in cancer growth and metastasis is the dissolution of the extracellular matrix surrounding the malignant tumor, which leads to tumor cell invasion and dissemination. Type I collagen degradation involves the initial action of collagenolytic matrix metalloproteinases (MMP-1, -8, and -13) activated by MMP-3 (stromelysin-1). The role of interactive matrix serine proteinases (MSPs), including tumor-associated trypsinogens, has been unclear in collagenolysis. Now, we provide evidence that the major isoenzyme of human tumor-associated trypsinogens, trypsin-2, can directly activate three collagenolytic proMMPs as well as proMMP-3. These proMMP activations are inhibited by tumor-associated trypsin inhibitor (TATI). Furthermore, we demonstrate that trypsin-2 efficiently degrades native soluble type I collagen, which can be inhibited by TATI. However, cell culture studies showed that trypsin-2 transfection into the HSC-3 cell line did not result in MMP-1, -3, -8, and -13 activation but affected MMP-3 and -8 production at the protein level. These findings indicate that human trypsin-2 can be regarded as a potent tumor-associated matrix serine protease capable of being the initial activator of the collagenolytic MMP activation network as well as directly attacking type I collagen.     

Nereis cuticle collagen. Proteolysis by marine vibrial and clostridial collagenases

Proteolysis of Nereis cuticle collagen by two bacterial collagenases was investigated using viscosimetry, enzyme kinetics, sodium dodecyl sulfate polyacrylamide gel electrophoresis, and ion exchange chromatography of collagenolytic peptides. Collagenase of the marine Vibrio B-30 completely degrades native cuticle collagen at 7 degress C with a turnover number 50 times greater than that of the clostridial collagenase. Although turnover numbers for the two enzymes are comparable when using denatured cuticle collagen as substrate, the vibrial collagenase appears to cleave twice as many peptide bonds per mg of cuticle collagen as does the clostridial enzyme. Sodium dodecyl sulfate gel electrophoresis of collagenase-digested native cuticle collagen reflects the resistance of the collagen to clostridial collagenase; however, the vibrial enzyme completely degrades the cuticle collagen with the formation of one transient intermediate (Mr 400,000). Peptide analysis of fully digested denatured cuticle collagen reveals that the two enzymes have a number of qualitative and quantitative similarities. Despite these, however, only the vibrial collagenase seems capable of extensively degrading native cuticle collagen.     

Collagenolytic proteases from bacteria

Collagen degradation occurs during various physiological and pathological conditions. However, only a limited number of proteases with unique characteristics can trigger collagen degradation. Until recently, practical applications of collagenolytic proteases from bacteria had not been considered because their functions in bacteria are closely related to pathogenicity. However, bacterial collagenolytic proteases have many interesting and useful features. This review focuses on the collagenolytic proteases from bacteria, in particular their molecular properties and practical applications.     

Matrix metalloproteinase-1 takes advantage of the induced fit mechanism to cleave the triple-helical type I collagen molecule

The collagenases are members of the matrix metalloproteinase family (MMP) that degrade native triple-helical type I collagen. To understand the mechanism by which these enzymes recognize and cleave this substrate, we studied the substrate specificity of a modified form of MMP-1 (FC) in which its active site region (amino acids 212-254) had been replaced with that of MMP-9 (amino acids 395-437). Although this substitution increased the activity of the enzyme toward gelatin and the peptide substrate Mca-PLGL(Dpa)AR-NH2 by approximately 3- and approximately 11-fold, respectively, it decreased the type I collagenolytic activity of the enzyme to 0.13%. The replacement of Gly233, the only amino acid in this region of FC that is conserved in all collagenase family members, with the corresponding Glu residue in MMP-9 resulted in a substantial decrease in the type I collagenolytic activity of the enzyme without affecting its general proteolytic activities. The kinetic parameters of the FC/G233E mutant for the collagen substrate were similar to those of the chimeric enzyme. In addition, substituting Gly233 for Glu in the chimera increased the collagenolytic activity of the enzyme by 12-fold. Interestingly, replacing Glu415 in MMP-9 with Gly, its corresponding residue in FC, endowed the enzyme with type I collagenolytic activity. The catalytic activity of the MMP-9 mutant toward triple-helical type I collagen was 2-fold higher than that of the collagenase chimera. These data in conjunction with the X-ray crystal structure of FC indicate that Gly233 provides the flexibility necessary for the enzyme active site to change conformation upon substrate binding. The flexibility provided by the Gly residue is essential for type I collagenolytic activity.     

Substrate specificity of the collagenolytic serine protease from Uca pugilator: studies with collagenous substrates

The collagenolytic protease from Uca pugilator was studied with respect to its catalytic properties on collagen types I-V. The crab protease degraded all five collagen types, producing multiple cleavages in the triple helix of each native collagen at 25 degrees C. The major early cleavage in the alpha 1 polypeptide chain of collagen types I-III occurred at a 3/4:1/4 locus, resulting in fragments electrophoretically similar to the TCA and TCB products of mammalian collagenase action. Interestingly, a propensity toward this same cleavage was observed even following thermal denaturation of the substrates. The ability of the crab protease to degrade all native collagen types and to catalyze cleavages at multiple loci in the triple helix distinguishes its action from that of mammalian collagenases. The collagenolytic activity of the crab protease was also examined on fibrillar collagen and compared to that of human skin fibroblast collagenase. Enzyme concentrations of fibroblast collagenase which resulted in the saturation of available substrate sites failed to show such an effect in the case of the crab protease. Binding studies of the crab protease to fibrillar collagen likewise indicated substantially reduced levels of enzyme binding in comparison to fibroblast collagenase. These data suggest that the affinity of the crab protease for native collagen is considerably less than the affinity of mammalian collagenase for this substrate.     

Purification and properties of individual collagenases fromStreptomyces sp. strain 3B

Streptomyces strain 3B constitutively secreted collagenolytic enzymes during the post-exponential growth phase. Purification is described here leading to two collagenases (I and II) with specific activity of 3350 and 3600 U/mg, respectively, the highest activity obtained as yet for any streptomycete collagenase. Analysis of the purified enzymes by the method of zymography revealed that both I and II were homogeneous, with molar mass 116 and 97 kDa, respectively. Both collagenases were identical in their pH (7.5) and temperature optimum (37 degrees C). The inhibition profile of the enzymes by EDTA and 1,10-phenanthroline confirmed these enzymes to be metalloproteinases. By testing the activity with insoluble collagen, acid soluble collagen, gelatin, casein, elastin and Pz-PLGPR it was established that I and II are very specific for insoluble collagen and gelatin, showing a high activity toward acid soluble collagen and Pz-PLGPR. However, collagenases I and II failed to hydrolyze casein and elastin; they belong to true collagenases and resemble the clostridial enzymes.